Martin Bilban

Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n−3 polyunsaturated fatty acids

Aims/hypothesisInflammatory alterations in white adipose tissue appear to underlie complications of obesity including diabetes mellitus. Polyunsaturated fatty acids (PUFA), particularly those of the n−3 series, modulate immune responses and may ameliorate insulin sensitivity. In this study, we investigated how PUFA affect white adipose tissue inflammation and gene expression in obese diabetic animals.Materials and methodsWe treated db/db mice as well as lean non-diabetic mice (db/+) with either low-fat standard diet (LF) or high-fat diets rich in (1) saturated/monounsaturated fatty acids (HF/S), (2) n−6 PUFA (HF/6) and (3) the latter including purified marine n−3 PUFA (HF/3).ResultsMany genes involved in inflammatory alterations were upregulated in db/db mice on HF/S compared with LF in parallel with phosphorylation of c-Jun N-terminal kinase (JNK). In parallel, adipose tissue infiltration with macrophages was markedly enhanced by HF/S. When compared with HF/S, HF/6 showed only marginal effects on adipose tissue inflammation. However, inclusion of n−3 PUFA in the diet (HF/3) completely prevented macrophage infiltration induced by high-fat diet and changes in inflammatory gene expression, also tending to reduce JNK phosphorylation (p<0.1) in diabetic mice despite unreduced body weight. Moreover, high-fat diets (HF/S, HF/6) downregulated expression and reduced serum concentrations of adiponectin, but this was not the case with n−3 PUFA.Conclusions/interpretationn−3 PUFA prevent adipose tissue inflammation induced by high-fat diet in obese diabetic mice, thereby dissecting obesity from adipose tissue inflammation. These data suggest that beneficial effects of n−3 PUFA on diabetes development could be mediated by their effect on adipose tissue inflammation.

Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)–deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

The sedoheptulose kinase CARKL directs macrophage polarization through control of glucose metabolism.

Summary Immune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.

Bilirubin A Natural Inhibitor of Vascular Smooth Muscle Cell Proliferation

Background—Bilirubin, a natural product of heme catabolism by heme oxygenases, was considered a toxic waste product until 1987, when its antioxidant potential was recognized. On the basis of observations that oxidative stress is a potent trigger in vascular proliferative responses, that heme oxygenase-1 is antiatherogenic, and that several studies now show that individuals with high-normal or supranormal levels of plasma bilirubin have a lesser incidence of atherosclerosis-related diseases, we hypothesized that bilirubin would have salutary effects on preventing intimal hyperplasia after balloon injury. Methods and Results—We found less balloon injury–induced neointima formation in hyperbilirubinemic Gunn rats and in wild-type rats treated with biliverdin, the precursor of bilirubin, than in controls. In vitro, bilirubin and biliverdin inhibited serum-driven smooth muscle cell cycle progression at the G1 phase via inhibition of the mitogen-activated protein kinase signal transduction pathways and inhibition of phosphorylation of the retinoblastoma tumor suppressor protein. Conclusions—Bilirubin and biliverdin might be potential therapeutics in vascular proliferative disorders.

Heme oxygenase and carbon monoxide initiate homeostatic signaling

Carbon monoxide (CO), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. Many biological functions of HO, such as regulation of vessel tone, smooth muscle cell proliferation, neurotransmission, and platelet aggregation, and anti-inflammatory and antiapoptotic effects have been attributed to its enzymatic product, CO. How can such diverse actions be achieved by a simple diatomic gas; can its protective effects be explained via regulation of a common signaling pathway? A number of the known signaling effects of CO depend on stimulation of soluble guanylate cyclase and/or activation of mitogen-activated protein kinases. The consequences of this activation remain unknown but appear to differ depending on cell type and circumstances. The majority of studies reporting a protective role of CO focus on pathways initiated by the pathological stimulus (e.g., lipopolysaccharide, hypoxia, balloon injury, tumor necrosis factor α, etc.) and its consequential modulation by CO. What has been less studied is the manner in which CO exposure alone modulates the molecular machinery of the cell so that a subsequent stress stimulus will elicit a homeostatic response as opposed to one that is chaotic and disordered. CO potentially interacts with other intracellular hemoprotein targets, although little is known about the functional significance of such interactions other then the known targets including mitochondrial oxidases, oxygen sensors, and nitric oxide synthases. The earliest response of a cell exposed to low concentrations of CO is clearly an increase in reactive oxygen species formation that we define as oxidative conditioning. This has important consequences for inflammation, proliferation, mitochondria biogenesis, and apoptosis. Within this review, we will highlight recent research on the molecular events underlying the physiologic effects of CO—which lead to cytoprotective conditioning.

Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver

Carbon monoxide (CO) has recently emerged as having potent cytoprotective properties; the mechanisms underlying these effects, however, are just beginning to be elucidated. In a rat model of lipopolysaccharide (LPS)‐induced multiorgan failure, we demonstrate that exposure to a low concentration of CO for only 1 h imparts a potent defense against lethal endotoxemia and effectively abrogates the inflammatory response. Exposure to CO leads to long‐term survival of >80% of animals vs. 20% in controls. In the lung, CO suppressed LPS‐induced lung alveolitis and associated edema formation, while in the liver, it reduced expression of serum alanine aminotransferase, a marker of liver injury. This protection appears to be based in part on different mechanisms in the lung and liver in that CO had reciprocal effects on LPS‐induced expression of iNOS and NO production, important mediators in the response to LPS. CO prevented the up‐regulation of iNOS and NO in the lung while augmenting expression of iNOS and NO in the liver. Studies of primary lung macrophages and hepatocytes in vitro revealed a similar effect; CO inhibited LPS‐induced cytokine production in lung macrophages while reducing LPS‐induced iNOS expression and nitrite accumulation and protected hepatocytes from apoptosis while augmenting iNOS expression. Although it is unclear to which extent these changes in iNOS contribute to the cytoprotection conferred by CO, it is fascinating that in each organ CO influences iNOS in a manner known to be protective in that organ: NO is therapeutic in the liver while it is damaging in the lung.

Identification of Heme Oxygenase-1 As a Novel BCR/ABL-Dependent Survival Factor in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL promotes the survival of leukemic cells. Heme oxygenase-1 (HO-1) is an inducible stress protein that catalyzes the degradation of heme and has recently been implicated in the regulation of growth and survival of various neoplastic cells. In the present study, we analyzed the expression and role of HO-1 in CML cells. As assessed by Northern and Western blot analysis as well as immunostaining, primary CML cells were found to express HO-1 mRNA and the HO-1 protein in a constitutive manner. Exposure of these cells to the BCR/ABL tyrosine kinase inhibitor STI571 resulted in decreased expression of HO-1 mRNA and protein. In addition, BCR/ABL was found to up-regulate HO-1 promoter activity, mRNA levels, and protein levels in Ba/F3 cells. To investigate the role of HO-1 for survival of primary CML cells, the HO-1 inducer hemin was used. Hemin-induced expression of HO-1 was found to protect CML cells from STI571-induced cell death. In addition, inhibition of HO-1 by zinc-(II)-deuteroporphyrin-IX-2,4-bisethyleneglycol resulted in a substantial decrease of cell viability. Furthermore, overexpression of HO-1 in the CML-derived cell line K562 was found to counteract STI571-induced apoptosis. Together, our data identify HO-1 as a novel BCR/ABL-driven survival molecule and potential target in leukemic cells in patients with CML. The pathogenetic and clinical implications of this observation remain to be elucidated.

The nuclear corepressor, NCoR, regulates thyroid hormone action in vivo

The thyroid hormone receptor (TR) has been proposed to regulate expression of target genes in the absence of triiodothyronine (T3) through the recruitment of the corepressors, NCoR and SMRT. Thus, NCoR and SMRT may play an essential role in thyroid hormone action, although this has never been tested in vivo. To accomplish this, we developed mice that express in the liver a mutant NCoR protein (L-NCoRΔID) that cannot interact with the TR. L-NCoRΔID mice appear grossly normal, however, when made hypothyroid the repression of many positively regulated T3-target genes is abrogated, demonstrating that NCoR plays a specific and sufficient role in repression by TR in the absence of T3. Remarkably, in the euthyroid state, expression of many T3-targets is also up-regulated in L-NCoRΔID mice, demonstrating that NCoR also determines the magnitude of the response to T3 in euthyroid animals. Although positive T3 targets were up-regulated in L-NCoRΔID mice in the hypo- and euthyroid state, there was little effect seen on negatively regulated T3 target genes. Thus, NCoR is a specific regulator of T3-action in vivo and mediates repression by the unliganded TR in hypothyroidism. Furthermore, NCoR appears to play a key role in determining the tissue-specific responses to similar levels of circulating T3. Interestingly, NCoR recruitment to LXR is also impaired in this model, leading to activation of LXR-target genes, further demonstrating that NCoR recruitment regulates multiple nuclear receptor signaling pathways.

Stat3 Is a Negative Regulator of Intestinal Tumor Progression in ApcMin Mice

BACKGROUND AND AIMS The transcription factor signal transducer and activator of transcription 3 (Stat3) has been considered to promote progression and metastasis of intestinal cancers. METHODS We investigated the role of Stat3 in intestinal tumors using mice with conditional ablation of Stat3 in intestinal epithelial cells (Stat3(DeltaIEC)). RESULTS In the Apc(Min) mouse model of intestinal cancer, genetic ablation of Stat3 reduced the multiplicity of early adenomas. However, loss of Stat3 promoted tumor progression at later stages, leading to formation of invasive carcinomas, which significantly shortened the lifespan of Stat3(DeltaIEC)Apc(Min/+) mice. Interestingly, loss of Stat3 in tumors of Apc(Min/+) mice had no significant impact on cell survival and angiogenesis, but promoted cell proliferation. A genome-wide expression analysis of Stat3-deficient tumors suggested that Stat3 might negatively regulate intestinal cancer progression via the cell adhesion molecule CEACAM1. CONCLUSIONS Our data suggest that Stat3 impairs invasiveness of intestinal tumors. Therefore, therapeutic targeting of the Stat3 signaling pathway in intestinal cancer should be evaluated for adverse effects on tumor progression.

Trophoblast invasion: Assessment of cellular models using gene expression signatures

Invasive, extravillous trophoblasts (EVT) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular markers with their primary counterpart, it is unknown to what extent they recapitulate the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs). Analyses of approximately 14,000 commonly expressed genes revealed that EVTs most closely resemble CTBs with considerable differences to the group of choriocarcinoma cells (JEG-3, BeWo, ACH-3P) and the group of SV40 Large T Antigen-selected cell types (SGHPL-5, HTR-8/SVneo). Similarly, analyses of 912 genes discriminating EVT from CTB, or 370 EVT-specific genes did not unravel a particular cell line with close similarity to any of the primary cell types, although molecular signatures common to EVT and each group of cell lines could be identified. Considering the diversity of mRNA expression patterns it is suggested that molecular studies in trophoblast cell lines require verification of the critical steps in an appropriate primary model system.